The long-term objective of this proposal is to elucidate mechanisms responsible for the establishment and maintenance of the polarized epithelial phenotype. Epithelial cells sort plasma membrane proteins and lipids into apical and basolateral domains in order to carry out vectorial functions for the organism. The experiments proposed will utilize the well-characterized MDCK cell system, established polarity assays and quantitative live imaging techniques to investigate fundamental aspects of the sorting of apical and basolateral plasma membrane proteins and the structure of the Trans Golgi Network (TGN). Specific aim 1 is to characterize in detail the exit pathways from the TGN of plasma membrane proteins with different apical and basolateral sorting signals. GFP-, YFP-, CFP- tagged proteins will be used in double and triple imaging assays, together with similarly tagged Golgi markers to determine the number, kinetics of Golgi exit, type of transporting intermediates and microtubule motor requirements of individual exit pathways from the Golgi complex. Specific aim 2 is to characterize in detail the role of adaptor protein complexes (AP1A, AP1 B, AP3, [unreadable] AP4, GGAs) and coat proteins in basolateral and apical sorting in TGN and endosomes. We will use [unreadable] biochemical targeting and recycling assays, antisense oligonucleotides, RNAi, specific inhibitors and imaging techniques to investigate the participation of these molecules in distinct apical and basolateral exit pathways from the TGN. Specific aim 3 is to study the mechanisms involved in the selective regulation of Golgi exit pathways and surface polarity by cdc42 in MDCK cells, recently demonstrated by our laboratory. We will search for downstream effectors of cdc42 that mediate these selective trafficking and polarity alterations using mutant forms of cdc42 selectively impaired in their ability to interact with downstream effectors (IQGAP, WASP, etc). The experiments proposed will provide novel information on the molecular bases of normal and abnormal function of epithelial organs, such as kidney, liver, intestine, lung and exocrine / endocrine glands. [unreadable] [unreadable]